Mitogenic oxygenase regulators

ABSTRACT

The present invention relates to new genes encoding for the production of novel nox enzyme proteins involved in generation of reactive oxygen intermediates that affect cell division. The present invention also provides vectors containing these genes, cells transfected with these vectors, antibodies raised against these novel proteins, kits for detection, localization and measurement of these genes and proteins, and methods to determine the activity of drugs to affect the activity of the proteins of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.09/999,248 filed Nov. 15, 2001 now U.S. Pat. No. 6,846,672 which claimspriority to U.S. Provisional Patent Application Ser. Nos. 60/249,305filed Nov. 16, 2000, 60/251,364 filed Dec. 5, 2000, 60/289,172 filed May7, 2001, and 60/289,537 filed May 7, 2001.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of NationalInstitutes of Health grants HL38206, HL58000, and CA84138.

TECHNICAL FIELD

The present invention relates to the field of normal and abnormal cellgrowth, in particular mitogenic regulation. The present inventionprovides the following: nucleotide sequences encoding for the productionof enzymes that are mitogenic regulators; amino acid sequences of theseenzymes; vectors containing these nucleotide sequences; methods fortransfecting cells with vectors that produce these enzymes; transfectedcells; methods for administering these transfected cells to animals toinduce tumor formation; antibodies to these enzymes that are useful fordetecting and measuring levels of these enzymes, and for binding tocells possessing extracellular epitopes of these enzymes; and assays forscreening for effectors of these enzymes.

BACKGROUND OF THE INVENTION

Reactive oxygen intermediates (ROI) are cytotoxic and mutagenic. ROIsmodify and damage critical biomolecules including DNA and lipids. Theare partial reduction products of oxygen: 1 electron reduces O₂ to formsuperoxide (O₂ ⁻), and 2 electrons reduce O₂ to form hydrogen peroxide(H₂O₂). The cytotoxic property of ROI is exploited by phagocytes, whichgenerate large amounts of superoxide and hydrogen peroxide as part oftheir armory of bactericidal mechanisms. ROI have been considered anaccidental byproduct of metabolism, particularly mitochondrialrespiration. Recent studies give evidence for regulated enzymaticgeneration of O₂ ⁻ and its conversion to H₂O₂ in a variety of cells. Theconversion of O₂ ⁻ to H₂O₂ can also occur spontaneously, but is markedlyaccelerated by superoxide dismutase (SOD). Exposure of cells to plateletderived growth factor and epidermal growth factor induces the productionof H₂O₂, which activates components of signaling pathways includingp42/p44 MAPK and tyrosine phosphroylation.

Several biological systems generate reactive oxygen. Exposure ofneutrophils to bacteria or to various soluble mediators such asformyl-Met-Leu-Phe or phorbol esters activates a massive consumption ofoxygen, termed the respiratory burst, to initially generate superoxide,with secondary generation of H₂O₂, HOCl and hydroxyl radical. The enzymeresponsible for this oxygen consumption is the respiratory burst oxidase(nicotinamide adenine dinucleotide phosphate-reduced form (NADPH)oxidase).

There is also growing evidence for the generation of ROI bynon-phagocytic cells, particularly in situations related to cellproliferation. Significant generation of H₂O₂, O₂ ⁻, or both have beennoted in some cell types. Fibroblasts and human endothelial cells showincreased release of superoxide in response to cytokines such asinterleukin-1 or tumor necrosis factor (TNF) (Meier et al. (1989)Biochem J. 263, 539–545.; Matsubara et al. (1986) J. Immun. 137,3295–3298). Ras-transformed fibroblasts show increased superoxiderelease compared with control fibroblasts (Irani, et al. (1997) Science275, 1649–1652). Rat vascular smooth muscle cells show increased H₂O₂release in response to PDGF (Sundaresan et al. (1995) Science 270,296–299) and angiotensin II (Griendling et al. (1994) Circ. Res. 74,1141–1148; Fukui et al. (1997) Circ. Res. 80, 45–51; Ushio-Fukai et al.(1996) J. Biol. Chem. 271, 23317–23321), and H₂O₂ in these cells isassociated with increased proliferation rate. H₂O₂ in the transformedfibroblasts and in vascular smooth muscle cells is associated with anincreased proliferation rate. The occurrence of ROI in a variety of celltypes is summarized in Table 1 (adapted from Burdon, R. (1995) FreeRadical Biol. Med. 18, 775–794).

TABLE 1 Superoxide Hydrogen Peroxide human fibroblasts Balb/3T3 cellshuman endothelial cells rat pancreatic islet cells human/rat smoothmuscle cells murine keratinocytes human fat cells rabbit chondrocyteshuman osteocytes human tumor cells BHK-21 cells fat cells, 3T3 L1 cellshuman colonic epithelial cells

ROI generated by neutrophils have a cytotoxic function. While ROI arenormally directed at the invading microbe, ROI can also induce tissuedamage (e.g., in inflammatory conditions such as arthritis, shock, lungdisease, and inflammatory bowel disease) or may be involved in tumorinitiation or promotion, due to damaging effects on DNA. Nathan(Szatrowski et al. (1991) Canc. Res. 51, 794–798) proposed that thegeneration of ROI in tumor cells may contribute to the hypermutabilityseen in tumors, and may therefore contribute to tumor heterogeneity,invasion and metastasis.

In addition to cytotoxic and mutagenic roles, ROI have ideal propertiesas signal molecules: 1) they are generated in a controlled manner inresponse to upstream signals; 2) the signal can be terminated by rapidmetabolism of O₂ ⁻ and H₂O₂ by SOD and catalase/peroxidases; 3) theyelicit downstream effects on target molecules, e.g., redox-sensitiveregulatory proteins such as NFκ-B and AP-1 (Schreck et al. (1991) EMBOJ. 10, 2247–2258; Schmidt et al. (1995) Chemistry & Biology 2, 13–22).Oxidants such as O₂ ⁻ and H₂O₂ have a relatively well defined signalingrole in bacteria, operating via the SoxI/II regulon to regulatetranscription.

ROI appear to have a direct role in regulating cell division, and mayfunction as mitogenic signals in pathological conditions related togrowth. These conditions include cancer and cardiovascular disease. O₂ ⁻is generated in endothelial cells in response to cytokines, and mightplay a role in angiogenesis (Matsubara et al. (1986) J. Immun. 137,3295–3298). O₂ ⁻ and H₂O₂ are also proposed to function as“life-signals”, preventing cells from undergoing apoptosis (Matsubara etal. (1986) J. Immun. 137, 3295–3298). As discussed above, many cellsrespond to growth factors (e.g., platelet derived growth factor (PDGF),epidermal derived growth factor (EGF), angiotensin II, and variouscytokines) with both increased production of O₂ ⁻/H₂O₂ and increasedproliferation. Inhibition of ROI generation prevents the mitogenicresponse. Exposure to exogenously generated O₂ ⁻ and H₂O₂ results in anincrease in cell proliferation. A partial list of responsive cell typesis shown below in Table 2 (adapted from Burdon, R. (1995) Free RadicalBiol. Med. 18, 775–794).

TABLE 2 Superoxide Hydrogen peroxide human, hamster fibroblasts mouseosteoblastic cells Balb/3T3 cells Balb/3T3 cells human histiocyticleukemia rat, hamster fibroblasts mouse epidermal cells human smoothmuscle cells rat colonic epithelial cells rat vascular smooth musclecells rat vascular smooth muscle cells

While non-transformed cells can respond to growth factors and cytokineswith the production of ROI, tumor cells appear to produce ROI in anuncontrolled manner. A series of human tumor cells produced largeamounts of hydrogen peroxide compared with non-tumor cells (Szatrowskiet al. (1991) Canc. Res. 51, 794–798). Ras-transformed NIH 3T3 cellsgenerated elevated amounts of superoxide, and inhibition of superoxidegeneration by several mechanisms resulted in a reversion to a “normal”growth phenotype.

O₂ ⁻ has been implicated in maintenance of the transformed phenotype incancer cells including melanoma, breast carcinoma, fibrosarcoma, andvirally transformed tumor cells. Decreased levels of the manganese formof SOD (MnSOD) have been measured in cancer cells and invitro-transformed cell lines, predicting increased O₂ ⁻ levels (Burdon,R. (1995) Free Radical Biol. Med. 18, 775–794). MnSOD is encoded onchromosome 6q25 which is very often lost in melanoma. Overexpression ofMnSOD in melanoma and other cancer cells (Church et al. (1993) Proc. ofNatl. Acad. Sci. 90, 3113–3117; Fernandez-Pol et al. (1982) Canc. Res.42, 609–617; Yan et al. (1996) Canc. Res. 56, 2864–2871) resulted insuppression of the transformed phenotype.

ROI are implicated in the growth of vascular smooth muscle associatedwith hypertension, atherosclerosis, and restenosis after angioplasty. O₂⁻ generation is seen in rabbit aortic adventitia (Pagano et al. (1997)Proc. Natl. Acad. Sci. 94, 14483–14488). Vascular endothelial cellsrelease O₂ ⁻ in response to cytokines (Matsubara et al. (1986) J. Immun.137, 3295–3298). O₂ ⁻ is generated by aortic smooth muscle cells inculture, and increased O₂ ⁻ generation is stimulated by angiotensin IIwhich also induces cell hypertrophy. In a rat model system, infusion ofangiotensin II leads to hypertension as well as increased O₂ ⁻generation in subsequently isolated aortic tissue (Ushio-Fukai et al.(1996) J. Biol. Chem. 271, 23317–23321.; Yu et al. (1997) J. Biol. Chem.272, 27288–27294). Intravenous infusion of a form of SOD that localizesto the vasculature or an infusion of an O₂ ⁻ scavenger preventedangiotensin II induced hypertension and inhibited ROI generation (Fukuiet al. (1997) Circ. Res. 80, 45–51).

The neutrophil NADPH oxidase, also known as phagocyte respiratory burstoxidase, provides a paradigm for the study of the specialized enzymaticROI-generating system. This extensively studied enzyme oxidizes NADPHand reduces oxygen to form O₂ ⁻. NADPH oxidase consists of multipleproteins and is regulated by assembly of cytosolic and membranecomponents. The catalytic moiety consists of flavocytochrome b₅₅₈, anintegral plasma membrane enzyme comprised of two components: gp91phox(gp refers to glycoprotein; phox is an abbreviation of the wordsphagocyte and oxidase) and p22phox (p refers to protein). gp91phoxcontains 1 flavin adenine dinucleotide (FAD) and 2 hemes as well as theNADPH binding site. p22phox has a C-terminal proline-rich sequence whichserves as a binding site for cytosolic regulatory proteins. The twocytochrome subunits, gp91 phox and p22phox appear to stabilize oneanother, since the genetic absence of either subunit, as in theinherited disorder chronic granulomatous disease (CGD), results in theabsence of the partner subunit (Yu et al. (1997) J. Biol. Chem. 272,27288–27294). Essential cytosolic proteins include p47phox, p67phox andthe small GTPase Rac, of which there are two isoforms. p47phox andp67phox both contain SH₃ regions and proline-rich regions whichparticipate in protein interactions governing assembly of the oxidasecomponents during activation. The neutrophil enzyme is regulated inresponse to bacterial phagocytosis or chemotactic signals byphosphorylation of p47phox, and perhaps other components, as well as byguanine nucleotide exchange to activate the GTP-binding protein Rac.

The origin of ROI in non-phagocytic tissues is unproven, but theoccurrence of phagocyte oxidase components has been evaluated in severalsystems by immunochemical methods, Northern blots and reversetranscriptase-polymerase chain reaction (RT-PCR). The message forp22phox is expressed widely, as is that for Rac1. Several cell typesthat are capable of O₂ ⁻ generation have been demonstrated to containall of the phox components including gp91phox, as summarized below inTable 3. These cell types include endothelial cells, aortic adventitiaand lymphocytes.

TABLE 3 Tissue gp91phox p22phox p47phox p67phox neutrophil +^(1,2)+^(1,2) +^(1,2) +^(1,2) aortic adventitia +¹ +¹ +¹ +¹ lymphocytes +² +²+^(1,2) +^(1,2) endothelial cells +² +² +^(1,2) +^(1,2) glomerularmesangial − +^(1,2) +^(1,2) +^(1,2) cells fibroblasts − +² +^(1,2) +²aortic sm. muscle − +^(1,2) ? ? ¹= protein expression shown. ²= mRNAexpression shown.

However, a distinctly different pattern is seen in several other celltypes shown in Table 3 including glomerular mesangial cells, rat aorticsmooth muscle and fibroblasts. In these cells, expression of gp91phox isabsent while p22phox and in some cases cytosolic phox components havebeen demonstrated to be present. Since gp91phox and p22phox stabilizeone another in the neutrophil, there has been much speculation that somemolecule, possibly related to gp91phox, accounts for ROI generation inglomerular mesangial cells, rat aortic smooth muscle and fibroblasts(Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317–23321).Investigation of fibroblasts from a patient with a genetic absence ofgp91phox provides proof that the gp91phox subunit is not involved in ROIgeneration in these cells (Emmendorffer et al. (1993) Eur. J. Haematol.51, 223–227). Depletion of p22phox from vascular smooth muscle using anantisense approach indicated that this subunit participates in ROIgeneration in these cells, despite the absence of detectable gp91phox(Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317–23321). At thistime the molecular candidates possibly related to gp91phox and involvedin ROI generation in these cells are unknown.

Accordingly, what is needed is the identity of the proteins involved inROI generation, particularly in non-phagocytic tissues and cells. Whatis also needed are the nucleotide sequences encoding for these proteins,and the primary sequences of the proteins themselves. Also needed arevectors designed to include nucleotides encoding for these proteins.Probes and PCR primers derived from the nucleotide sequence are neededto detect, localize and measure nucleotide sequences, including mRNA,involved in the synthesis of these proteins. In addition, what is neededis a means to transfect cells with these vectors. What is also neededare expression systems for production of these molecules. Also neededare antibodies directed against these molecules for a variety of usesincluding localization, detection, measurement and passive immunization.

SUMMARY OF THE INVENTION

The present invention solves the problems described above by providing anovel family of nucleotide sequences and proteins, termed Nox proteins,encoded by these nucleotide sequences. In particular the presentinvention provides compositions comprising the nucleotide sequences SEQID NO:1, SEQ ID NO:3, or SEQ ID NO:5, and conservative substitutions andfragments thereof, wherein SEQ ID NO:1 and fragments thereof code forthe expression of the protein comprising SEQ ID NO:2 and fragmentsthereof; and SEQ ID NO:3 and fragments thereof code for the expressionof the protein comprising SEQ ID NO:4 and fragments thereof. SEQ ID NO:5is the promoter sequence for Nox 1. While not wanting to be bound by thefollowing statement, it is believed that these Nox proteins, SEQ ID NOs:2 and 4, and fragments thereof, are involved in ROI production. Thepresent invention also provides vectors containing these nucleotidesequences, cells transfected with these vectors which produce theproteins comprising SEQ ID NO:2 and SEQ ID NO:4, or fragments thereof,and antibodies to these proteins and fragments thereof. The presentinvention also provides methods for stimulating cellular proliferationby administering vectors encoded for production of the proteinscomprising SEQ ID NO:2 or SEQ ID NO:4, and fragments thereof. Thepresent invention further provides methods for stimulating cellularproliferation by administering the proteins comprising SEQ ID NO:2 orSEQ ID NO:4, and fragments thereof. The nucleotides and antibodies ofthe present invention are useful for the detection, localization andmeasurement of the nucleic acids encoding for the production of theproteins of the present invention, and also for the detection,localization and measurement of the proteins of the present invention.These nucleotides and antibodies can be combined with other reagents inkits for the purposes of detection, localization and measurement.

Most particularly, the present invention involves a method forregulation of cell division or cell proliferation by modifying theactivity or expression of the proteins described as SEQ ID NO:2 or SEQID NO:4, or fragments thereof. These proteins, in their naturallyoccurring or expressed forms, are expected to be useful in drugdevelopment, for example for screening of chemical and drug libraries byobserving inhibition of the activity of these enzymes. Such chemicalsand drugs would likely be useful as treatments for cancer, prostatichypertrophy, benign prostatic hypertrophy, hypertension, atherosclerosisand many other disorders involving abnormal cell growth or proliferationas described below. The entire expressed protein may be useful in theseassays. Portions of the molecule which may be targets for inhibition ormodification include, but are not limited to, the binding site forpyridine nucleotides (NADPH or NADH), the flavoprotein domain(approximately the C-terminal 265 amino acids), and/or the binding orcatalytic site for flavin adenine dinucleotide (FAD).

The present invention further comprises the creation ofreporter-promoter constructs for use in assays to measure the activityof compounds. The method of the present invention may additionally beused for the development of drugs or other therapies for the treatmentof conditions associated with abnormal growth including, but not limitedto, cancer, psoriasis, prostatic hypertrophy, benign prostatichypertrophy, cardiovascular disease, proliferation of vessels, includingbut not limited to blood vessels and lymphatic vessels, arteriovenousmalformation, vascular problems associated with the eye,atherosclerosis, hypertension, and restenosis following angioplasty. Theenzymes of the present invention are excellent targets for thedevelopment of drugs and other agents which may modulate the activity ofthese enzymes. It is to be understood that modulation of activity mayresult in enhanced, diminished or absence of enzymatic activity.Modulation of the activity of these enzymes may be useful in treatmentof conditions associated with abnormal growth.

Drugs which affect the activity of the enzymes represented in SEQ IDNO:2, SEQ ID NO:4, or fragments thereof, may also be combined with othertherapeutics in the treatment of specific conditions. For example, thesedrugs may be combined with angiogenesis inhibitors in the treatment ofcancer, with antihypertensives for the treatment of hypertension, andwith cholesterol lowering drugs for the treatment of atherosclerosis.

Accordingly, an object of the present invention is to provide nucleotidesequences, or fragments thereof, encoding for the production ofproteins, or fragments thereof, that are involved in ROI production.

Another object of the present invention is to provide vectors containingthese nucleotide sequences, or fragments thereof.

Yet another object of the present invention is to provide cellstransfected with these vectors.

Still another object of the present invention is to administer cellstransfected with these vectors to animals and humans.

Another object of the present invention is to provide proteins, orfragments thereof, that are involved in ROI production.

Still another object of the present invention is to provide antibodies,including monoclonal and polyclonal antibodies, or fragments thereof,raised against proteins, or fragments thereof, that are involved in ROIproduction.

Another object of the present invention is to administer genescontaining nucleotide sequences, or fragments thereof, encoding for theproduction of proteins, or fragments thereof, that are involved in ROIproduction, to animals and humans, and also to cells obtained fromanimals and humans.

Another object of the present invention is to administer antisensecomplimentary sequences of genes containing nucleotide sequences, orfragments thereof, encoding for the production of proteins, or fragmentsthereof, that are involved in ROI production, to animals and humans andalso to cells obtained from animals and humans.

Yet another object of the present invention is to provide a method forstimulating or inhibiting cellular proliferation by administeringvectors containing nucleotide sequences, or fragments thereof, encodingfor the production of proteins, or fragments thereof, that are involvedin ROI production, to animals and humans. It is also an object of thepresent invention to provide a method for stimulating or inhibitingcellular proliferation by administering vectors containing antisensecomplimentary sequences of nucleotide sequences, or fragments thereof,encoding for the production of proteins, or fragments thereof, that areinvolved in ROI production, to animals and humans. These methods ofstimulating cellular proliferation are useful for a variety of purposes,including but not limited to, developing animal models of tumorformation, stimulating cellular proliferation of blood marrow cellsfollowing chemotherapy or radiation, or in cases of anemia.

Still another object of the present invention is to provide antibodiesuseful in immunotherapy against cancers expressing the proteinsrepresented in SEQ ID NO:2, SEQ ID NO:4, or fragments thereof.

Yet another object of the present invention is to provide nucleotideprobes useful for the detection, localization and measurement ofnucleotide sequences, or fragments thereof, encoding for the productionof proteins, or fragments thereof, that are involved in ROI production.

Another object of the present invention is to provide antibodies usefulfor the detection, localization and measurement of nucleotide sequences,or fragments thereof, encoding for the production of proteins, orfragments thereof, that are involved in ROI production.

Another object of the present invention is to provide kits useful fordetection of nucleic acids including the nucleic acids including thenucleic acids represented in SEQ ID NO:1, SEQ ID NO:3, or fragmentsthereof, that encode for proteins, or fragments thereof, that areinvolved in ROI production.

A further object of the present invention is to provide kits useful fordetection of nucleic acids including nucleic acids represented in SEQ IDNO:5, or fragments thereof, representing the promoter region of Nox 1.

Still another object of the present invention is to provide kits usefulfor the localization of nucleic acids including the nucleic acidsrepresented in SEQ ID NO:1, SEQ ID NO:3, or fragments thereof, thatencode for proteins, or fragments thereof that are involved in ROIproduction.

Yet another object of the present invention is to provide kits usefulfor the localization of nucleic acids including the nucleic acidsrepresented in SEQ ID NO:5 or fragments thereof, representing thepromoter region of Nox 1.

Another object of the present invention is to provide kits useful fordetection of proteins, including the proteins represented in SEQ ID NO:2and SEQ ID NO:4, or fragments thereof, that are involved in ROIproduction.

Yet another object of the present invention is to provide kits usefulfor detection and measurement of proteins, including the proteinsrepresented in SEQ ID NO:2 and SEQ ID NO:4, or fragments thereof, thatare involved in ROI production.

Still another object of the present invention is to provide kits usefulfor localization of proteins, including the proteins represented in SEQID NO:2 and SEQ ID NO:4, or fragments thereof, that are involved in ROIproduction.

Yet another object of the present invention is to provide kits usefulfor the detection, measurement or localization of nucleic acids, orfragments thereof, encoding for proteins, or fragments thereof, that areinvolved in ROI production, for use in diagnosis and prognosis ofabnormal cellular proliferation related to ROI production.

Another object of the present invention is to provide kits useful forthe detection, measurement or localization of proteins, or fragmentsthereof, that are involved in ROI production, for use in diagnosis andprognosis of abnormal cellular proliferation related to ROI production.

A further object of the present invention is to use the proteinsrepresented in SEQ ID NO:2 and SEQ ID NO:4, or fragments thereof, toscreen for drugs that regulate the cellular levels or activity ofproteins in the Nox family.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a dendrogram indicating the degree of similarity among thisfamily of proteins, and also includes the related plant enzymes.

FIG. 2( a–b) depicts the alignment of the predicted protein sequences ofgp91phox, Nox 1, Nox 3, Nox 4 and Nox 5.

FIG. 3 is a model consistent with the known features of gp91phox.

FIG. 4( a–b) depicts tissue expression of Nox 3, Nox 4, and Nox 5measured by Northern Blot analysis.

FIG. 5 shows RT-PCR measurement of tissue expression of the Nox familyof proteins. RT-PCR was carried out using Nox-specific PCR primers asdescribed herein. The number of cycles was 35, except where indicated(number of cycles in parentheses).

FIG. 6( a–b) depicts expression of Nox isoforms in tumor or transformedcell lines; FIG. 6c shows the ratio of expression of gp91phox, Nox 4,and Nox 5 compared with glyceraldehyde-3-phosphate dehydrogenase(G3PDH), obtained from real time PCR results.

FIG. 7 depicts the creation of a promoter-reporter construct for Nox 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention solves the problems described above by providing anovel family of nucleotide sequences, and proteins termed Nox proteins,encoded by these nucleotide sequences. The term “Nox” refers to“NADPH-oxidase.” These novel proteins are part of a larger relatedfamily of proteins that generate ROI, including mox proteins (mox is anabbreviation for mitogenic NADPH oxidase), and Duox proteins, (duox isan abbreviation for dual oxidase). In particular, the present inventionprovides novel compositions comprising the nucleotide sequences SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5, and fragments thereof. SEQ ID NO:1,or fragments thereof, encode for proteins comprising SEQ ID NO:2 orfragments thereof. SEQ ID NO:3, or fragments thereof, encode forproteins comprising SEQ ID NO:4 or fragments thereof. SEQ ID NO:5 is thepromoter region for Nox 1.

The Nox proteins described herein have homology to the gp91phox proteininvolved in ROI generation, however, the Nox proteins comprise a noveland distinct family of proteins. The Nox proteins included in thepresent invention have a molecular weight of approximately 65 kDa asdetermined by reducing gel electrophoresis and are capable of inducingROI generation in cells. As described in detail below, the Nox proteinsof the present invention also function in the regulation of cell growth,and are therefore implicated in diseases involving abnormal cell growthsuch as cancer. The present invention describes Nox proteins found inhumans, however, it is likely that the Nox family of genes/proteins iswidely distributed among multicellular organisms.

In addition to the nucleotide sequences described above, the presentinvention also provides vectors containing these nucleotide sequencesand fragments thereof, cells transfected with these vectors whichproduce the proteins comprising SEQ ID NO:2, SEQ ID NO:4, and fragmentsthereof, and antibodies to these proteins and fragments thereof. Thepresent invention also provides methods for stimulating cellularproliferation by administering vectors, or cells containing vectors,encoded for production of the proteins comprising SEQ ID NO:2, SEQ IDNO:4, and fragments thereof. The nucleotides and antibodies of thepresent invention are useful for the detection, localization andmeasurement of the nucleic acids encoding for the production of theproteins of the present invention, and also for the detection,localization and measurement of the proteins of the present invention.These nucleotides and antibodies can be combined with other reagents inkits for the purposes of detection, localization and measurement. Thesekits are useful for diagnosis and prognosis of conditions involvingcellular proliferation associated with production of reactive oxygenintermediates.

The present invention solves the problems described above by providing acomposition comprising the nucleotide sequence SEQ ID NO:1 and fragmentsthereof. The present invention also provides a composition comprisingthe nucleotide sequence SEQ ID NO:3 and fragments thereof. The presentinvention additionally provides a composition comprising the nucleotidesequence SEQ ID NO: 5 and fragments thereof.

The present invention provides a composition comprising the protein SEQID NO:2 encoded by the nucleotide sequence SEQ ID NO:1. The presentinvention additionally provides a composition comprising the protein SEQID NO:4 encoded by the nucleotide sequence SEQ ID NO:3.

The present invention provides a composition comprising the protein SEQID NO:2 or fragments thereof, encoded by the nucleotide sequence SEQ IDNO:1 or fragments thereof. The present invention also provides acomposition comprising the protein SEQ ID NO:4 or fragments thereof,encoded by the nucleotide sequence SEQ ID NO:3 or fragments thereof.

The present invention also provides vectors containing the nucleotidesequences SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, and fragmentsthereof. The present invention also provides cells transfected withthese vectors. In addition, the present invention provides cells stablytransfected with the nucleotide sequence SEQ ID NO:1 or fragmentsthereof. The present invention also provides cells stably transfectedwith the nucleotide sequence SEQ ID NO:3 or fragments thereof.

The present invention provides cells stably transfected with thenucleotide sequence SEQ ID NO:1 or fragments thereof, which produce theprotein SEQ ID NO:2 or fragments thereof. In addition, the presentinvention provides cells stably transfected with the nucleotide sequenceSEQ ID NO:3 or fragments thereof which produce the protein SEQ ID NO:4or fragments thereof.

The present invention provides a method for stimulating growth byadministering cells stably transfected with the nucleotide sequence SEQID NO:1 which produce the protein SEQ ID NO:2 or fragments thereof. Thepresent invention also provides a method for stimulating growth byadministering cells stably transfected with the nucleotide sequence SEQID NO:3 or fragments thereof, which produce the protein SEQ ID NO:4 orfragments thereof.

Specifically, the present invention provides a method for stimulatingtumor formation by administering cells stably transfected with thenucleotide sequence SEQ ID NO:1 or fragments thereof, which produce theprotein SEQ ID NO:2 or fragments thereof. The present invention alsoprovides a method for stimulating tumor formation by administering cellsstably transfected with the nucleotide sequence SEQ ID NO:3 or fragmentsthereof, which produce the protein SEQ ID NO:4 or fragments thereof.

The present invention may also be used to develop anti-sense nucleotidesequences to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or fragmentsthereof. These anti-sense molecules may be used to interfere withtranslation of nucleotide sequences, such as SEQ ID NO:1, or SEQ IDNO:3, or fragments thereof, that encode respectively, for proteins suchas SEQ ID NO:2, SEQ ID NO:4, or fragments thereof. Administration ofthese anti-sense molecules, or vectors encoding for these anti-sensemolecules, to humans and animals, would interfere with production ofproteins such as SEQ ID NO:2, SEQ ID NO:4, or fragments thereof, therebydecreasing production of ROIs and inhibiting cellular proliferation.These methods are useful in producing animal models for use in study oftumor development and vascular growth, and for study of the efficacy oftreatments for affecting tumor and vascular growth in vivo.

The present invention also provides a method for high throughputscreening of drugs and chemicals which modulate the proliferativeactivity of the enzymes of the present invention, thereby affecting celldivision. Combinatorial chemical libraries may be screened for chemicalswhich modulate the proliferative activity of these enzymes. Drugs andchemicals may be evaluated based on their ability to modulate theenzymatic activity of the expressed or endogenous proteins, includingthose represented by SEQ ID NO:2, SEQ ID NO:4, or fragments thereof.Endogenous proteins may be obtained from many different tissues orcells, such as colon cells. Drugs may also be evaluated based on theirability to bind to the expressed or endogenous proteins represented bySEQ ID NO:2, SEQ ID NO:4, or fragments thereof. Enzymatic activity maybe NADPH- or NADH-dependent superoxide generation catalyzed by theholoprotein. Enzymatic activity may also be NADPH- or NADH-dependentdiaphorase activity catalyzed by either the holoprotein or theflavoprotein domain.

By flavoprotein domain is meant approximately the C-terminal half of theenzymes shown in SEQ ID NO:2, SEQ ID NO:4, or fragments thereof. Theseproteins and fragments thereof have NADPH-dependent reductase activitytowards cytochrome c, nitrobluetetrazolium and other dyes. Expressedproteins or fragments thereof can be used for robotic screens ofexisting combinatorial chemical libraries. While not wanting to be boundby the following statement, it is believed that the NADPH or NADHbinding site and the FAD binding site are useful for evaluating theability of drugs and other compositions to bind to the Nox enzymes or tomodulate their enzymatic activity. The use of the holoprotein or theC-terminal half or end regions are preferred for developing a highthroughput drug screen.

The present invention also provides antibodies directed to the proteinsSEQ ID NO:2, SEQ ID NO:4, and fragments thereof. The antibodies of thepresent invention are useful for a variety of purposes includinglocalization, detection and measurement of the proteins SEQ ID NO:2, SEQID NO:4, and fragments thereof. The antibodies may be employed in kitsto accomplish these purposes. These antibodies may also be linked tocytotoxic agents for selected killing of cells. The term antibody ismeant to include any class of antibody such as IgG, IgM and otherclasses. The term antibody also includes a completely intact antibodyand also fragments thereof, including but not limited to Fab fragmentsand Fab+Fc fragments.

The present invention also provides the nucleotide sequences SEQ IDNO:1, SEQ ID NO:3, and fragments thereof. These nucleotide sequences areuseful for a variety of purposes including localization, detection, andmeasurement of messenger RNA involved in synthesis of the proteinsrepresented as SEQ ID NO:2, SEQ ID NO:4, and fragments thereof. Thepresent invention also provides the nucleotide sequence for SEQ ID NO:5and fragments thereof. This nucleotide sequence is useful for a varietyof purposes including localization, detection and measurement ofmessenger RNA involved in synthesis of the Nox family of proteins. Thesenucleotides may also be used in the construction of labeled probes forthe localization, detection, and measurement of nucleic acids such asmessenger RNA or alternatively for the isolation of larger nucleotidesequences containing the nucleotide sequences shown in SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, or fragments thereof. These nucleotide sequencesmay be used to isolate homologous strands from other species usingtechniques known to one of ordinary skill in the art. These nucleotidesequences may also be used to make probes and complementary strands.

Most particularly, the present invention involves a method formodulation of growth by modifying the proteins represented as SEQ IDNO:2, SEQ ID NO:4, or fragments thereof.

The term “mitogenic regulators” is used herein to mean any molecule thatacts to affect cell division.

The term “animal” is used herein to mean humans and non-human animals ofboth sexes.

The terms “a”, “an” and “the” as used herein are defined to mean one ormore and include the plural unless the context is inappropriate.“Proteins”, “peptides,” “polypeptides” and “oligopeptides” are chains ofamino acids (typically L-amino acids) whose alpha carbons are linkedthrough peptide bonds formed by a condensation reaction between thecarboxyl group of the alpha carbon of one amino acid and the amino groupof the alpha carbon of another amino acid. The terminal amino acid atone end of the chain (i.e., the amino terminal) has a free amino group,while the terminal amino acid at the other end of the chain (i.e., thecarboxy terminal) has a free carboxyl group. As such, the term “aminoterminus” (abbreviated N-terminus) refers to the free alpha-amino groupon the amino acid at the amino terminal of the protein, or to thealpha-amino group (imino group when participating in a peptide bond) ofan amino acid at any other location within the protein. Similarly, theterm “carboxy terminus” (abbreviated C-terminus) refers to the freecarboxyl group on the amino acid at the carboxy terminus of a protein,or to the carboxyl group of an amino acid at any other location withinthe protein.

Typically, the amino acids making up a protein are numbered in order,starting at the amino terminal and increasing in the direction towardthe carboxy terminal of the protein. Thus, when one amino acid is saidto “follow” another, that amino acid is positioned closer to the carboxyterminal of the protein than the preceding amino acid.

The term “residue” is used herein to refer to an amino acid (D or L) oran amino acid mimetic that is incorporated into a protein by an amidebond. As such, the amino acid may be a naturally occurring amino acidor, unless otherwise limited, may encompass known analogs of naturalamino acids that function in a manner similar to the naturally occurringamino acids (i.e., amino acid mimetics). Moreover, an amide bond mimeticincludes peptide backbone modifications well known to those skilled inthe art.

Furthermore, one of skill in the art will recognize that, as mentionedabove, individual substitutions, deletions or additions which alter, addor delete a single amino acid or a small percentage of amino acids (lessthan about 20%, typically less than about 10%, more typically less thanabout 1%) in an encoded sequence are conservatively modified variationswhere the alterations result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.The following six groups each contain amino acids that are conservativesubstitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

When the peptides are relatively short in length (i.e., less than about50 amino acids), they are often synthesized using standard chemicalpeptide synthesis techniques. Solid phase synthesis in which theC-terminal amino acid of the sequence is attached to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence is a preferred method for the chemical synthesis of theantigenic epitopes described herein. Techniques for solid phasesynthesis ate known to those skilled in the art.

Alternatively, the antigenic epitopes described herein are synthesizedusing recombinant nucleic acid methodology. Generally, this involvescreating a nucleic acid sequence that encodes the peptide or protein,placing the nucleic acid in an expression cassette under the control ofa particular promoter, expressing the peptide or protein in a host,isolating the expressed peptide or protein and, if required, renaturingthe peptide or protein. Techniques sufficient to guide one of skillthrough such procedures are found in the literature.

When several desired protein fragments or peptides are encoded in thenucleotide sequence incorporated into a vector, one of skill in the artwill appreciate that the protein fragments or peptides may be separatedby a spacer molecule such as, for example, a peptide, consisting of oneor more amino acids. Generally, the spacer will have no specificbiological activity other than to join the desired protein fragments orpeptides together, or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of thespacer may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity. Nucleotide sequencesencoding for the production of residues which may be useful inpurification of the expressed recombinant protein may be built into thevector. Such sequences are known in the art. For example, a nucleotidesequence encoding for a poly histidine sequence may be added to a vectorto facilitate purification of the expressed recombinant protein on anickel column.

Once expressed, recombinant peptides, polypeptides and proteins can bepurified according to standard procedures known to one of ordinary skillin the art, including ammonium sulfate precipitation, affinity columns,column chromatography, gel electrophoresis and the like. Substantiallypure compositions of about 50 to 99% homogeneity are preferred, and 80to 95% or greater homogeneity are most preferred for use as therapeuticagents.

One of skill in the art will recognize that after chemical synthesis,biological expression or purification, the desired proteins, fragmentsthereof and peptides may possess a conformation substantially differentthan the native conformations of the proteins, fragments thereof andpeptides. In this case, it is often necessary to denature and reduceprotein and then to cause the protein to re-fold into the preferredconformation. Methods of reducing and denaturing proteins and inducingre-folding are well known to those of skill in the art.

The genetic constructs of the present invention include coding sequencesfor different proteins, fragments thereof, and peptides. The geneticconstructs also include epitopes or domains chosen to permitpurification or detection of the expressed protein. Such epitopes ordomains include DNA sequences encoding the glutathione binding domainfrom glutathione S-transferase, hexa-histidine, thioredoxin,hemagglutinin antigen, maltose binding protein, and others commonlyknown to one of skill in the art. The preferred genetic constructincludes the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:5, or fragments thereof. It is to be understood that additional oralternative nucleotide sequences may be included in the geneticconstructs in order to encode for the following: a) multiple copies ofthe desired proteins, fragments thereof, or peptides; b) variouscombinations of the desired proteins, fragments thereof, or peptides;and c) conservative modifications of the desired proteins, fragmentsthereof, or peptides, and combinations thereof. Preferred proteinsinclude the human Nox 4 protein and human Nox 5 protein shown as SEQ IDNO:2 and SEQ ID NO:4, respectively, and fragments thereof orconservative substitutions thereof.

The nucleotide sequences of the present invention may also be employedto hybridize to nucleic acids such as DNA or RNA nucleotide sequencesunder high stringency conditions which permit detection, for example, ofalternately spliced messages.

The genetic construct is expressed in an expression system such as inNIH 3T3 cells using recombinant sequences in a pcDNA-3 vector(Invitrogen, Carlsbad, Calif.) to produce a recombinant protein.Preferred expression systems include but are not limited to Cos-7 cells,insect cells using recombinant baculovirus, and yeast. It is to beunderstood that other expression systems known to one of skill in theart may be used for expression of the genetic constructs of the presentinvention. The preferred proteins of the present invention are thesequences referred to herein as human Nox 4 and human Nox 5 or fragmentsthereof which have the amino acid sequences set forth in SEQ ID NO:2 andSEQ ID NO:4, respectively, or an amino acid sequence having amino acidsubstitutions as defined in the definitions that do not significantlyalter the function of the recombinant protein in an adverse manner.

Terminology

It should be understood that some of the terminology used to describethe novel Nox proteins contained herein is different from theterminology in PCT/US99/26592, U.S. non-provisional application Ser. No.09/437,568 and U.S. provisional application Ser. Nos. 60/251,364,60/249,305, and 60/289,172. The terms mox and nox are equivalents. Asdescribed herein, the term “human Nox 4” refers to a protein comprisingan amino acid sequence as set forth in SEQ ID NO:2, or fragments orconservative substitutions thereof, and encoded by the nucleotidesequence as set forth in SEQ ID NO:1, or fragments or conservativesubstitutions thereof. As described herein, the term “human Nox 5”refers to a protein comprising an amino acid sequence as set forth inSEQ ID NO:4, or fragments or conservative substitutions thereof, andencoded by the nucleotide sequence as set forth in SEQ ID NO:3, orfragments or conservative substitutions thereof. The promoter for “humanNox 1” refers to a nucleic acid sequence as set forth in SEQ ID NO:5 orfragments or conservative substitutions thereof.

Construction of the Recombinant Gene

The desired gene is ligated into a transfer vector, such as pcDNA3, andthe recombinants are used to transform host cells such as Cos-7 cells.It is to be understood that different transfer vectors, host cells, andtransfection methods may be employed as commonly known to one ofordinary skill in the art. Three desired genes for use in transfectionare shown in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. For example,lipofectamine-mediated transfection and in vivo homologous recombinationwas used to introduce the Nox 4 gene (SEQ ID NO:1) into NIH 3T3 cells.

The synthetic gene is cloned and the recombinant construct containing aNox gene is produced and grown in confluent monolayer cultures of aCos-7 cell line. The expressed recombinant protein is then purified,preferably using affinity chromatography techniques, and its purity andspecificity determined by known methods.

A variety of expression systems may be employed for expression of therecombinant protein. Such expression methods include, but are notlimited to the following: bacterial expression systems, including thoseutilizing E. coli and Bacillus subtilis; virus systems; yeast expressionsystems; cultured insect and mammalian cells; and other expressionsystems known to one of ordinary skill in the art.

Transfection of Cells

It is to be understood that the vectors of the present invention may betransfected into any desired cell or cell line. Both in vivo and invitro transfection of cells are contemplated as part of the presentinvention. Preferred cells for transfection include but are not limitedto the following: fibroblasts (possibly to enhance wound healing andskin formation), granulocytes (possible benefit to increase function ina compromised immune system as seen in AIDS, and aplastic anemia),muscle cells, neuroblasts, stem cells, bone marrow cells, osteoblasts, Blymphocytes, and T lymphocytes.

Cells may be transfected with a variety of methods known to one ofordinary skill in the art and include but are not limited to thefollowing: electroporation, gene gun, calcium phosphate, lipofectamine,and fugene, as well as adenoviral transfection systems.

Host cells transfected with the nucleic acids represented in SEQ IDNO:1, SEQ ID NO:3, or fragments thereof, are used to express theproteins SEQ ID NO:2, SEQ ID NO:4, respectively, or fragments thereof.Host cells transfected with the nucleic acid represented in SEQ ID NO:1,SEQ ID NO:3, or SEQ ID NO:5 or fragments thereof, are also used asscreening assays.

These expressed proteins are used to raise antibodies. These antibodiesmay be used for a variety of applications including but not limited toimmunotherapy against cancers expressing one of the Nox proteins, andfor detection, localization and measurement of the proteins shown in SEQID NO:2, SEQ ID NO:4, or fragments thereof.

Purification and Characterization of the Expressed Protein

The proteins of the present invention can be expressed as a fusionprotein with a poly histidine component, such as a hexa histidine, andpurified by binding to a metal affinity column using nickel or cobaltaffinity matrices. The protein can also be expressed as a fusion proteinwith glutathione S-transferase and purified by affinity chromatographyusing a glutathione agarose matrix. The protein can also be purified byimmunoaffinity chromatography by expressing it as a fusion protein, forexample with hemagglutinin antigen. The expressed or naturally occurringprotein can also be purified by conventional chromatographic andpurification methods which include anion and cation exchangechromatography, gel exclusion chromatography, hydroxylapatitechromatography, dye binding chromatography, ammonium sulfateprecipitation, precipitation in organic solvents or other techniquescommonly known to one of skill in the art.

Methods of Assessing Activity of Expressed Proteins

Different methods are available for assessing the activity of theexpressed proteins of the present invention, including but not limitedto the proteins represented as SEQ ID NO:2, SEQ ID NO:4, conservativesubstitutions thereof, and fragments thereof.

1. Assays of the Holoprotein and Fragments Thereof for SuperoxideGeneration

A. General Considerations.

These assays are useful in assessing efficacy of drugs designed tomodulate the activity of the enzymes of the present invention. Theholoprotein may be expressed in COS-7 cells, NIH 3T3 cells, insect cells(using baculoviral technology) or other cells using methods known to oneof skill in the art. Membrane fractions or purified protein are used forthe assay. The assay may require or be augmented by other cellularproteins such as p47phox, p67phox, and Rac1, as well as potentiallyother unidentified factors (e.g., kinases or other regulatory proteins).

B. Cytochrome c Reduction.

NADPH or NADH is used as the reducing substrate, in a concentration ofabout 100 μM. Reduction of cytochrome c is monitoredspectrophotometrically by the increase in absorbance at 550 nm, assumingan extinction coefficient of 21 mM⁻¹cm⁻¹. The assay is performed in theabsence and presence of about 10 μg superoxide dismutase. Thesuperoxide-dependent reduction is defined as cytochrome c reduction inthe absence of superoxide dismutase minus that in the presence ofsuperoxide dismutase (Uhlinger et al. (1991) J. Biol. Chem. 266,20990–20997). Acetylated cytochrome c may also be used, since thereduction of acetylated cytochrome c is thought to be exclusively viasuperoxide.

C. Nitroblue Tetrazolium Reduction.

For nitroblue tetrazolium (NBT) reduction, the same general protocol isused, except that NBT is used in place of cytochrome c. In general,about 1 mL of filtered 0.25% nitrotetrazolium blue (Sigma, St. Louis,Mo.) is added in Hanks buffer without or with about 600 Units ofsuperoxide dismutase (Sigma) and samples are incubated at approximately37° C. The oxidized NBT is clear, while the reduced NBT is blue andinsoluble. The insoluble product is collected by centrifugation, and thepellet is re-suspended in about 1 mL of pyridine (Sigma) and heated forabout 10 minutes at 100° C. to solubilize the reduced NBT. Theconcentration of reduced NBT is determined by measuring the absorbanceat 510 nm, using an extinction coefficient of 11,000 M⁻¹cm⁻¹. Untreatedwells are used to determine cell number.

D. Luminescence.

Superoxide generation may also be monitored with a chemiluminescencedetection system utilizing lucigenin (bis-N-methylacridinium nitrate,Sigma, St. Louis, Mo.). The sample is mixed with about 100 μM NADPH(Sigma, St. Louis, Mo.) and 10 μM lucigenin (Sigma, St. Louis, Mo.) in avolume of about 150 μL Hanks solution. Luminescence is monitored in a96-well plate using a LumiCounter (Packard, Downers Grove, Ill.) for 0.5second per reading at approximately 1 minute intervals for a total ofabout 5 minutes; the highest stable value in each data set is used forcomparisons. As above, superoxide dismutase is added to some samples toprove that the luminescence arises from superoxide. A buffer blank issubtracted from each reading (Ushio-Fukai et al. (1996) J. Biol. Chem.271, 23317–23321).

E. Assays in Intact Cells.

Assays for superoxide generation may be performed using intact cells,for example, the Nox-transfected NIH 3T3 cells. In principle, any of theabove assays can be used to evaluate superoxide generation using intactcells, for example, the Nox-transfected NIH 3T3 cells. NBT reduction isa preferred assay method.

2. Assays of Truncated Proteins Comprised of Approximately theC-terminal 265 Amino Acid Residues

While not wanting to be bound by the following statement, the truncatedprotein comprised of approximately the C-terminal 265 amino acidresidues is not expected to generate superoxide, and therefore,superoxide dismutase is not added in assays of the truncated protein.Basically, a similar assay is established and the superoxide-independentreduction of NBT, cytochrome c, dichlorophenolindophenol, ferricyanide,or another redox-active dye is examined.

Nucleotides and Nucleic Acid Probes

The nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, as wellas fragments thereof and PCR primers therefore, may be used,respectively, for localization, detection and measurement of nucleicacids related to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, as well asfragments thereof. The nucleotide sequences SEQ ID NO:1 and SEQ ID NO:3are also called the human Nox 4 gene and the human Nox 5 generespectively, in this application. The nucleotide sequence SEQ ID NO:5is called the Nox 1 promoter sequence in this application.

The nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, as wellas fragments and conservative substitutions thereof, may be used tocreate probes to isolate larger nucleotide sequences containing thenucleotide sequences SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5,respectively. The nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5 as well as fragments thereof and conservative substitutionsthereof, may also be used to create probes to identify and isolate Noxproteins in other species.

The nucleic acids described herein include messenger RNA coding forproduction of SEQ ID NO:2, SEQ ID NO:4, and fragments and conservativesubstitutions thereof. Such nucleic acids include but are not limited tocDNA probes. These probes may be labeled in a variety of ways known toone of ordinary skill in the art. Such methods include but are notlimited to isotopic and non-isotopic labeling. These probes may be usedfor in situ hybridization for localization of nucleic acids such as mRNAencoding for SEQ ID NO:2, SEQ ID NO:4, and fragments and conservativesubstitutions thereof. Localization may be performed using in situhybridization at both ultrastructural and light microscopic levels ofresolution using techniques known to one of ordinary skill in the art.

These probes may also be employed to detect and quantitate nucleic acidsand mRNA levels using techniques known to one of ordinary skill in theart including but not limited to solution hybridization.

Administration of the Nox Proteins of the Present Invention

The proteins represented by SEQ ID NO:2, or SEQ ID NO:4, or fragments orconservative substitutions thereof, are combined with a pharmaceuticallyacceptable carrier or vehicle to produce a pharmaceutical compositionand are administered to animals. Such administration may occur forstimulation of growth or cellular proliferation. Administration may alsooccur for generation of antibodies.

The terms “pharmaceutically acceptable carrier or pharmaceuticallyacceptable vehicle” are used herein to mean any liquid including but notlimited to water or saline, oil, gel, salve, solvent, diluent, fluidointment base, liposome, micelle, giant micelle, and the like, which issuitable for use in contact with living animal or human tissue withoutcausing adverse physiological responses, and which does not interactwith the other components of the composition in a deleterious manner.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletscommonly used by one of ordinary skill in the art.

Preferred unit dosage formulations are those containing a dose or unit,or an appropriate fraction thereof, of the administered ingredient. Itshould be understood that in addition to the ingredients, particularlymentioned above, the formulations of the present invention may includeother agents commonly used by one of ordinary skill in the art.

The pharmaceutical composition may be administered through differentroutes, such as oral, including buccal and sublingual, rectal,parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal,and topical. The pharmaceutical composition of the present invention maybe administered in different forms, including but not limited tosolutions, emulsions and suspensions, microspheres, particles,microparticles, nanoparticles, and liposomes.

The pharmaceutical composition may be stored at temperatures of fromabout 4° C to −100° C. The pharmaceutical composition may also be storedin a lyophilized state at different temperatures including roomtemperature. The pharmaceutical composition may be sterilized throughconventional means known to one of ordinary skill in the art. Such meansinclude, but are not limited to filtration, radiation and heat. Thepharmaceutical composition of the present invention may also be combinedwith bacteriostatic agents, such as thimerosal, to inhibit bacterialgrowth.

Administration may also occur for the production of polyclonalantibodies using methods known to one of ordinary skill in the art. Thepreferred animals for antibody production are rabbits and mice. Otheranimals may be employed for immunization with these proteins orfragments thereof. Such animals include, but are not limited to thefollowing; sheep, horses, pigs, donkeys, cows, monkeys and rodents suchas guinea pigs and rats. It is expected that from about 1 to 7 dosagesmay be required per immunization regimen. Initial injections may rangefrom about 0.1 μg to 1 mg, with a preferred range of about 1 μg to 800μg, and a more preferred range of from approximately 25 μg to 500 μg.Booster injections may range from 0.1 μg to 1 mg, with a preferred rangeof approximately 1 μg to 800 μg, and a more preferred range of about 10μg to 500 μg.

The volume of administration will vary depending on the route ofadministration and the size of the recipient. For example, intramuscularinjections may range from about 0.1 ml to 1.0 ml.

Adjuvants

A variety of adjuvants known to one of ordinary skill in the art may beadministered in conjunction with the protein in the pharmaceuticalcomposition for generation of antibodies. Such adjuvants include, butare not limited to the following: polymers, co-polymers such aspolyoxyethylene-polyoxypropylene copolymers, including blockco-polymers; polymer P1005; Freund's complete adjuvant (for animals);Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300adjuvant; alum; QS 21, muramyl dipeptide; trehalose; bacterial extracts,including mycobacterial extracts; detoxified endotoxins; membranelipids; or combinations thereof.

Monoclonal antibodies can be produced using hybridoma technology inaccordance with methods well known to those skilled in the art. Theantibodies are useful as research or diagnostic reagents or can be usedfor passive immunization. The composition may optionally contain anadjuvant.

The polyclonal and monoclonal antibodies useful as research ordiagnostic reagents may be employed for detection and measurement of SEQID NO:2, SEQ ID NO:4, and fragments or conservative substitutionsthereof. Such antibodies may be used to detect these proteins in abiological sample, including but not limited to samples such as cells,cellular extracts, tissues, tissue extracts, biopsies, tumors, andbiological fluids. Such detection capability is useful for detection ofdisease related to these proteins to facilitate diagnosis and prognosisand to suggest possible treatment alternatives.

Detection may be achieved through the use of immunocytochemistry, ELISA,radioimmunoassay or other assays as commonly known to one of ordinaryskill in the art. The Nox 4 and Nox 5 proteins, or fragments orconservative substitutions thereof, may be labeled through commonlyknown approaches, including but not limited to the following:radiolabeling, dyes, magnetic particles, biotin-avidin, fluorescentmolecules, chemiluminescent molecules and systems, ferritin, colloidalgold, and other methods known to one of skill in the art of labelingproteins.

Administration of Antibodies

The antibodies directed to the proteins shown as SEQ ID NO:2, SEQ IDNO:4, or directed to fragments or conservative substitutions thereof,may also be administered directly to humans and animals in a passiveimmunization paradigm. Antibodies directed to extracellular portions ofSEQ ID. NO:2; SEQ ID NO:4, or fragments thereof bind to theseextracellular epitopes. Attachment of labels to these antibodiesfacilitates localization and visualization of sites of binding.Attachment of molecules such as ricin or other cytotoxins to theseantibodies helps to selectively damage or kill cells expressing SEQ IDNO:2, SEQ ID NO:4, or fragments thereof.

Kits

The present invention includes kits useful with the antibodies, nucleicacid probes, labeled antibodies, labeled proteins or fragments thereoffor detection, localization and measurement of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or combinations thereof andfragments or conservative substitutions thereof. The diagnostic kits mayalso measure or detect the relative expression of the Nox proteinsdescribed herein (i.e. human Nox 4 and/or human Nox 5)

Kits may be used for immunocytochemistry, in situ hybridization,solution hybridization, radioimmunoassay, ELISA, Western blots,quantitative PCR, and other assays for the detection, localization andmeasurement of these nucleic acids, proteins or fragments thereof usingtechniques known to one of skill in the art.

The nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,or fragments thereof, may also be used under high stringency conditionsto detect alternately spliced messages related to SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, or fragments thereof, respectively.

Fragments of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, containing therelevant hybridizing sequence can be synthesized onto the surface of achip array. RNA samples, e.g., from tumors, are then fluorescentlytagged and hybridized onto the chip for detection. This approach may beused diagnostically to characterize tumor types and to tailor treatmentsand/or provide prognostic information. Such prognostic information mayhave predictive value concerning disease progression and life span, andmay also affect choice of therapy.

The other present invention is further illustrated by the followingexamples, which are not to be construed in any way as imposinglimitations upon the scope thereof. On the contrary, it is to be clearlyunderstood that resort may be had to various other embodiments,modifications, and equivalents thereof, which, after reading thedescription herein, may suggest themselves to those skilled in the artwithout departing from the spirit of the present invention.

EXAMPLE 1

Sequence Analysis and Cloning of the Human Nox 4 cDNA (SEQ ID NO:1)Encoding for Production of the Human Nox 4 Protein (SEQ ID NO:2)

Using Nox 3 (SEQ ID NO:6) as a query sequence, a 789 base pair sequencedportion of expressed sequence tag (EST) (SEQ ID NO: 27) and a 408 baseEST clone SEQ ID NO: 28, a clone exhibiting a 26% identity to the cDNAsequence corresponding to amino acid residues 433–560 of Nox 3, and asecond clone showing 36% identity to the cDNA sequence corresponding toamino acid residues 5–48 of Nox 3 were discovered. This homologue wascloned using two PCR primers based on the two EST sequences: (SEQ IDNO:7, 5′-CAACGAAGGGGTTAAACACCTCTGC-3′; and SEQ ID NO:8,5′-CACAGCTGATTGATTCCGCTGAG-3′). PCR was carried out using human fetalkidney marathon-ready cDNA (Clontech, Palo Alto, Calif.), and the 0.85kb product was sequenced. Based on the sequencing results, 5′- and3′-RACE using the same library using the following primers: 5′-RACE: SEQID NO:9, 5′-TAAGCCAAGAGTGTTCGGCACATG-3′; SEQ ID NO:10,5′-TACTCTGGCCCTTGGTTATACAGCA-3′(for nested PCR); 3′-RACE: SEQ ID NO:11,5′-TCCATTTACCCTCACAATGTGT-3′; SEQ ID NO:12,5′-CTCAGCGGAATCAATCAGCTGTG-3′(for nested PCR) was then carried out. PCRparameters were 95° C. for 30 s, 62° C. or 65° C. for 20 s, 72° C. for45 s 25–35 cycles as indicated after denaturing for 1 min 30 s at 95° C.PCR products were purified with a QIAquick PCR purification kit or a gelpurification kit (QIAGEN, Valencia, Calif.). The positive PCR bands weresequenced by ABI 377 automatic sequencing. Primers were designed tosubclone the full-length cDNA and the correct sequence was confirmed byautomated sequencing.

Secretion signal sequences were predicted according to web-based SMARTprogram (version 3.1) at EMBL (Heidelberg, Germany). Prediction of openreading frames (ORF) was carried out using the EditSeq program(DNASTAR), and phylogenetic analyses and multiple sequence alignmentwere carried out using the clustal method using the Megalign program(DNASTAR). Transmembrane alpha helices were predicted using the TMHMMalgorithms through the Center for Biological Sequence Analysis (Lyngby,Denmark).

Total RNA was extracted from cell lines with Trizol (Life Technologies,Gaithersburg, Md.) based on the manufacturer's protocol or according to(Ishii et al., 1999) for glioma cell lines. RNAs were reversetranscribed into first-strand cDNA With Superscript II (LifeTechnologies, Gaithersburg, Md.) using oligo-dT according on the methodprovided by the manufacturer.

Table 4 shows the basic features of the cDNA and the predicted proteins.Like the proteins encoded by gp91phox (a.k.a., Nox 2 SEQ ID NO:13) andNox 1 (SEQ ID NO:14), the new sequences encode predicted proteins ofaround 65 kDa, and message sizes are similar in length (2.0–2.2 kb). Nox4 also has 59 amino acids which are strongly basic, 45 amino acids whichare strongly acidic, 212 hydrophobic amino acids, 171 polar amino acids,an isoelectic point of 8.695 and a charge of 16.549 at a pH of 7.0. Nox4 also shows 21–59% identity with gp91phox and with Nox 1. Nox 1,gp91phox, Nox 3 and Nox 4 cluster within a sub-family that is similar togp91phox. The alignment of the predicted protein sequence of Nox 4 isshown in FIG. 2. The molecules are roughly divided into two largedomains: an N-terminal cluster of hydrophobic membrane-spanningsequences, and a C-terminal flavoprotein domain. The latter shows weakhomology with a number of FAD binding proteins including cytochromeP-450 reductase and ferredoxin-NADP oxidoreductase (Rotrosen et al.,1992; Segal et al., 1992). Within the flavoprotein domain are twosegments (indicated in FIG. 2( a–b)) that show homology with known FADbinding sites in other flavoproteins, and four segments closer to theC-terminus that are homologous to documented pyridine nucleotide bindingsites in other proteins. The first of these includes the G-X-G-X-X-Pcanonical sequence that characterizes pyridine nucleotide binding sites.In all Nox forms, this sequence is followed by an F, which is typical ofNADPH- rather than NADH-specific enzymes.

Nox 4 contains the predicted transmembrane alpha helix near the extremeN-terminus (light hashed box in FIG. 2). However, this region is alsostrongly predicted to be a signal peptide sequence in these forms.Predicted proteolytic cleavage sites for each isoform are indicated bythe arrows, and cleavage at these positions would lead to a loss of thefirst putative transmembrane sequence. Five additional transmembraneregions are also predicted in this protein. The most C-terminal of theseis weakly predicted in Nox 1, gp91phox, Nox 3 and Nox 4 and is entirelymissed by some prediction algorithms. It is necessary to include thistransmembrane region in order to generate a model (FIG. 3) which isconsistent with known features of gp91phox, particularly a cytosolicfacing location of the flavoprotein domain. In this model, knownN-linked glycosylation sites in gp91phox are correctly localized toextracellular loops (although these sites are not conserved in otherisoforms). In addition, a polybasic loop of gp91phox that binds to thecytosolic regulatory protein p47phox (Biberstine-Kinkade et al., 1999)is localized on the cytosolic face. In general, extracellular loops tendto be highly variable in length and sequence, whereas the transmembranehelices and intracellular loops tend to be more conserved in sequenceand length (FIG. 2).

Within the N-terminus are five absolutely conserved histidines (FIG. 2),that are also conserved in all other members of the Nox family ofenzymes. gp91phox contains two heme groups, the irons of which are eachligated by two histidyl nitrogens (Isogai et al., 1993), and these arethought to reside within the N-terminus (Yu et al., 1998). An additionalconserved histidine lies within the FAD-binding region and is thereforenot a candidate for heme ligation. Thus, four of the five histidineswithin the N-terminus probably participate in heme ligation, providingpart of the binding sites for two heme groups, as indicated in FIG. 3.

TABLE 4 Molecular Features of Nox 3, Nox 4, and Nox 5 cDNA Nox 3 Nox 4Nox 5 cDNA length (bp) 2044 2232 2199 Predicted number of 568 578 565amino acids Predicted protein Mw 64.9 66.9 64.7 (kDa) pI of protein 88.7 9.7 Kozak sequence ATCATGA or GGCATGG GTCATGG ATGATGG Identity togp91phox 58% 37% 27% Identity to NoxI 55% 35% 29% SEQ ID NO: 29  30  31 

EXAMPLE 2

Tissue Expression of Nox 4 mRNA

The predominant Nox 4 2.4 kb message, which corresponds to the sizeexpected for the full-length Nox 4 transcript, is highly expressed inadult as well as fetal kidney (FIG. 4A). An additional weak Nox 4 bandwas also detected at 4.5 kilobases (kb) in fetal and adult kidney (FIG.4A). It is particularly expressed at the site of erythropoietinproduction in the kidney. RT-PCR confirmed kidney expression and alsorevealed expression of Nox 4 in all fetal tissues tested as well as inseveral adult tissues including pancreas, placenta, ovary, testis andskeletal muscle.

EXAMPLE 3

Sequence Analysis and Cloning of the cDNA for Human Nox 5

The Blast search using Nox 3 (SEQ ID NO:6) as a query sequence alsoidentified homology with fragments of genomic clone RP11-809H16 ofchromosome 15, these fragments are SEQ ID NOS: 32, 33 and 34. Theseclones exhibit 46 to 50% identity to Nox 3 within three exons. 5′- and3′-RACE were carried out using human fetal kidney marathon-ready cDNA(Clontech, Palo Alto, Calif.), using the following four primers whichwere designed based on the genomic sequence: SEQ ID NO:15,5′-CTCATTGTCACACTCCTCGACAGC-3′; SEQ ID NO:16,5′-TGGGTCTGATGCCTTGAAGGACTC-3′(for nested PCR); 3′-RACE: SEQ ID NO:17,5′-ATCAAGCGGCCCCCTTTTTTTCAC-3′; SEQ ID NO:18,5′-CTGAACATCCCCACCATTGCTCGC-3′(for nested PCR). PCR parameters were 95°C. for 30 s, 62° C. or 65° C. for 20 s, 72° C. for 45 s, 25–35 cycles asindicated after denaturing for 1.5 minutes at 95° C. PCR products werepurified with a QIAquick PCR purification kit or a gel purification kit(QIAGEN, Valencia, Calif.). Primers were designed to subclone thefull-length cDNA and the correct sequence was confirmed by ABI 3777automated sequencing.

Secretion signal sequences were predicted according to web-based SMARTprogram (version 3.1) at EMBL (Heidelberg, Germany). Prediction of openreading frames (ORF) was carried out using the EditSeq program(DNASTAR), and phylogenetic analyses and multiple sequence alignmentwere carried out using the clustal method using the Megalign program(DNASTAR). Transmembrane alpha helices were predicted using the TMHMMalgorithms through the Center for Biological Sequence Analysis (Lyngby,Denmark).

Total RNA was extracted from cell lines with Trizol (Life Technologies,Gaithersburg, Md.) based on the manufacturer's protocol or according toIshii et al., (1999) for glioma cell lines. RNAs were reversetranscribed into first-strand cDNA with Superscript II (LifeTechnologies, Gaithersburg, Md.) using oligo-dT according on the methodprovided by the manufacturer.

Table 4 shows the basic features of the cDNA and the predicted proteins.Like the proteins encoded by gp91phox (SEQ ID NO:13) and Nox 1 (SEQ IDNO:14), the new sequences for Nox 4 and Nox 5 encode predicted proteinsof around 65 kDa, and message sizes are similar in length (2.0.–2.2 kb).Nox 3, Nox 4 and Nox 5 show 21–59% identity with gp91phox. Nox 5 forms aunique group, of which it is the only member identified to date, andwhich is highly divergent from other members of the family. Based on itsposition in the family tree, Nox 5 may represent the gene which isclosest to the primordial Nox.

The alignment of the predicted protein sequences of gp91phox, Nox 1, Nox3, Nox 4 and Nox 5 is shown in FIG. 2. The molecules are roughly dividedinto two large domains: an N-terminal cluster of hydrophobicmembrane-spanning sequences, and a C-terminal flavoprotein domain. Thelatter shows weak homology with a number of FAD binding proteinsincluding cytochrome P-450 reductase and ferredoxin-NADP oxidoreductase(Rotrosen et al., 1992; Segal et al., 1992). Within the flavoproteindomain are two segments (indicated in FIG. 2) that show homology withknown FAD binding sites in other flavoproteins, and four segments nearerthe C-terminus that are homologous to documented pyridine nucleotidebinding sites in other proteins. The first of these includes theG-X-G-X-X-P canonical sequence that characterizes pyridine nucleotidebinding sites. In all Nox forms, this sequence is followed by an F,which is typical of NADPH- rather than NADH-specific enzymes.

While the N-terminal half of Nox 1, Nox 3, Nox 4, and Nox 5 are allhydrophobic, Nox 5 differs from the others somewhat in the details ofpredicted transmembrane alpha helices, as illustrated in FIG. 2. Nox 5does not contain an N-terminal predicted signal peptide, but doescontain a predicted transmembrane alpha helix (first hashed box, Nox 5sequence in FIG. 2). According to the prediction algorithm, the extremeN-terminus of Nox 5 is located on the inside of the membrane, on thesame side as the flavoprotein domain. Five additional transmembraneregions are also predicted in these proteins. The most C-terminal ofthese is strongly predicted in Nox 5. It is necessary to include thistransmembrane region in order to generate a model (FIG. 3) which isconsistent with known features of gp91phox, particularly a cytosolicfacing location of the flavoprotein domain. In this model, knownN-linked glycosylation sites in gp91phox are correctly localized toextracellular loops (although these sites are not conserved in otherisoforms). In addition, a polybasic loop of gp91phox that binds to thecytosolic regulatory protein p47phox (Biberstine-Kinkade et al., 1999)is localized on the cytosolic face. In general, extracellular loops tendto be highly variable in length and sequence, whereas the transmembranehelices and intracellular loops tend to be more conserved in sequenceand length (FIG. 2).

Within the N-terminus are five absolutely conserved histidines (FIG. 2),that are also conserved in all other members of the Nox family ofenzymes (data not shown). gp91phox contains two heme groups, the ironsof which are each ligated by two histidyl nitrogens (Isogai et al.,1993), and these are thought to reside within the N-terminus (Yu et al.,1998). An additional conserved histidine lies within the FAD-bindingregion and is therefore not a candidate for heme ligation. Thus, four ofthe five histidines within the N-terminus probably participate in hemeligation, providing part of the binding sites for two heme groups, asindicated in FIG. 3.

Additionally, located at the extreme N-terminus on the cytosolic side ofthe membrane of Nox 5 is a highly cationic proline-rich sequence (thePro-Arg-Rich sequence indicated in FIG. 2 and FIG. 3). This region isthought to serve as a binding sequence for Src-Homology 3 (SH3) domainsin another protein. SH3 domains are known to recognize inter- orintra-molecular proline-rich sequences. This is similar to p22phox, amembrane-associated subunit that associates with gp91phox, and containsa C-terminal, proline-rich sequence (Parkos et al., 1988) that serves asa binding site for a SH3 domain in p47phox, one of the cytosolicsubunits that regulates the activity of gp91phox. Although not wantingto be bound by the following statement, it is possible that theproline-rich sequence in Nox 5 serves as an internal p22phox, allowinginteraction with cytosolic regulatory proteins.

EXAMPLE 4

Tissue Expression of Nox 5 mRNA

Northern blots probed for Nox 5 using a 3′-portion of the coding region(FIG. 4A) revealed the presence of a 2.2 kb band corresponding in sizeto the full-length Nox 5 transcript in all fetal tissues tested. Thisspecies was also seen in low amounts in adult spleen and testis, alongwith larger transcripts at 2.6 kb and 6 kb. A probe using a portion ofthe 3′ untranslated region also revealed the presence of the same 2.6 kband 6 kb bands (FIG. 4B). Thus, these larger bands are largertranscripts derived from the same gene. RT-PCR confirmed expression ofNox 5 in testis and spleen, and also revealed weak expression in ovary,placenta, and pancreas (FIG. 5).

EXAMPLE 5

Real Time RT-PCR of Nox 4 and Nox 5.

G3PDH was used as a control. The G3PDH PCR product was purified using aQIAquick PCR purification kit (QIAGEN, Valencia, Calif.) and quantifiedusing absorbance at 260 nm using a BECKMAN DU640B spectrophotometer. Thestandard curve for G3PDH was constructed using 10-fold serial dilutionsof a known concentration of G3PDH PCR product in distilled water. Realtime PCR amplification was carried out using a LightCycler (RocheMolecular Biochemicals, Indianapolis, Ind.) in a PCR reaction containing0.2 μM of each primer, 1:84,000 SYBR Green I (Molecular Probes, Eugene,Oreg.) and Advantage 2 Polymerase Mix (Clontech, Polo Alto, Calif.).Amplification was carried out for 36 cycles of denaturation (95° C., 0s, ramp rate 20°/s), annealing (65° C., 5 s, ramp rate 20/s) andextension (72° C., 30 s ramp rate 20° C./s). Fluorescence was monitoredat the end of each extension phase. Quantitation and melting curve wereanalyzed with the LightCycler software. RT-PCR confirmed kidneyexpression and also revealed expression of Nox 4 in all fetal tissuestested as well as in several adult tissues including pancreas, placenta,ovary, testis and skeletal muscle. (See FIG. 5) The ratio of copies ofunknown to standard G3PDH was then calculated and is reported in FIG.6C. RT-PCR also confirmed expression of Nox 5 in testis and spleen, andrevealed weak expression in ovary, placenta, and pancreas (FIG. 5). Thedata indicate that expression patterns of Nox family members are tissuespecific, and do not correspond to the expression of gp91phox.

EXAMPLE 6

Northern Blotting of Nox 4 and Nox 5

The Human Fetal and Adult Multiple Tissue Northern Blot (Clontech, PaloAlto, Calif.) was hybridized with ³²P-random primer-labeled Nox 4, orNox probe according to the manufacturer's instructions. The probes wereprepared by PCR with primers for Nox 4: SEQ ID NO:7 and SEQ ID NO:8; andprimers for Nox 5: SEQ ID NO:19, 5′-CTGAACATCCCCACCATTGCTCGC-3′ and SEQID NO: 20, 5′-GAAGCCGAACTTCTCACAATGGCC-3′. The PCR products representcoding sequences corresponding to amino acids 11–294 (Nox 4), or 278–557(Nox 5). Because the Nox 5 transcript sizes differ between fetal andadult northern blots, a 420 bp PCR product of the Nox 5 3′-untranslatedregion amplified by primers (SEQ ID NO: 215′-CCTCACCTCTCCAAGCTCTGCCCC-3′ and SEQ ID NO: 225′-TTGAACAATTTTATAAGATGCCGG-3′) was also used to hybridize NorthernBlots.

The predominant Nox 4 2.4 kb message, which corresponds to the sizeexpected for the full-length Nox 4 transcript, is highly expressed inadult as well as fetal kidney (FIG. 4A), confirming recent reports(Kikuchi et al., 2000; Geiszt et al., 2000; Shiose et al., 2000). Anadditional weak Nox 4 band was also detected at 4.5 kilobases (kb) infetal and adult kidney (FIG. 4A). Northern blots probed for Nox 5 (FIG.4A) revealed the presence of a 2.2 kb band corresponding in size to thefull-length Nox 5 transcript in all fetal tissues tested. This specieswas also seen in low amounts in adult spleen and testis, along withlarger transcripts at 2.6 kb and 6 kb. A probe using a portion of the 3′untranslated region also revealed the presence of the same 2.6 kb and 6kb bands (FIG. 4B). Thus, these larger bands are larger transcriptsderived from the same gene.

EXAMPLE 7

Transfection of NIH3T3 Cells with SEQ ID NO:1 or SEQ ID NO:3

The nucleotide sequence SEQ ID NO:1 or SEQ ID NO:3 encoding forproduction of the Nox 4 protein (SEQ ID NO:2) or the Nox 5 protein (SEQID NO:4), respectively, is subcloned into the Not1 site of the pEF-PACvector (obtained from Mary Dinauer, Indiana University Medical School,Indianapolis, Ind.) which has a puromycin resistance gene. Transfectionis carried out as described in Sambrook et al., Molecular Cloning, ALaboratory Manual, Volumes 1–3, 2nd edition, Cold Spring HarborLaboratory Press, N.Y., 1989. The SEQ ID NO:1 in pEF-PAC and the emptyvector are separately transfected into NIH 3T3 cells using Fugene 6(Boeringer Mannheim).

10⁵ to 10³ cells stably transfected separately with human Nox 4 gene SEQID NO:1, with human Nox 5 gene SEQ ID NO:3, and with empty vector areprepared in 0.3% warm (40° C.) agar solution containing DMEM and 10%calf serum. Cells are distributed onto a hardened 0.6% agar plateprepared with DMEM and 10% calf serum. After three weeks in culture (37°C., 5% CO₂) colony formation is observed by microscopy.

About 2×10⁶ cells maintained in DMEM containing 10% calf serum aretransfected with 10 μg of DNA. After 2 days, cells are split andselected in the same medium containing 1 mg/ml puromycin. Colonies thatsurvive in selection media for 10 to 14 days are subculturedcontinuously in the presence of puromycin.

Cells which are stably transfected with the empty vector and cultured insoft agar for 3 weeks as above do not display anchorage independentgrowth. In contrast, NIH 3T3 cells which are stably transfected with theNox 4 (SEQ ID NO:1) or with the Nox 5 gene (SEQ ID NO:3) cultured for 3weeks in soft agar demonstrate anchorage independent growth of colonies.Transfected cells exhibit a transformed-like morphology, similar to thatseen with (V12)Ras-transfected cells, characterized by long spindle-likecells.

EXAMPLE 8

Expression of Nox 4 (SEQ ID NO:1) or Nox 5 (SEQ ID NO:3) in TransfectedNIH3T3 Cells

To verify the expression of Nox 4 mRNA or Nox 5 mRNA after transfection,RT-PCR and Northern blotting are performed. Total RNAs are prepared from10⁶ cells using the High Pure RNA Isolation Kit (Boeringer Mannheim) orRneasy kit (Qiagen). cDNAs, for each colony are prepared from 1–2 μg oftotal RNA using Advantage RT-PCR Kit (ClonTech). PCR amplification isperformed using primers, SEQ ID NO: 23 and SEQ ID NO:24. For Northernblotting, 10–20 μg of total RNA is separated on a 1% agaroseformaldehyde gel and transferred to a nylon filter. After ultraviolet(UV) cross-linking, filters are used for Northern blotting assay asdescribed in Example 6. Colonies expressing large amounts of Nox 4 mRNAor Nox 5 mRNA are chosen for further analysis.

EXAMPLE 9

NADPH-Dependent Superoxide Generation Assay

In one embodiment of the present invention, NIH 3T3 cells stablytransfected with the human Nox 4 gene (SEQ ID NO:1) or human Nox 5 gene(SEQ ID NO:3) are analyzed for superoxide generation using the lucigenin(Bis-N-methylacridinium luminescence assay (Sigma, St. Louis, Mo., Li etal. (1998) J. Biol. Chem. 273, 2015–2023). Cells are washed with coldHANKS' solution and homogenized on ice in HANKS' buffer containing 15%sucrose using a Dounnce homogenizer. Cell lysates are frozen immediatelyin a dry ice/ethanol bath. For the assay, 30 μg of cell lysate is mixedwith 200 μM NADPH and 500 μM lucigenin. Luminescence is monitored usinga LumiCounter (Packard) at three successive one minute intervals and thehighest value was used for comparison. Protein concentration isdetermined by the Bradford method.

Superoxide generation is monitored in lysates from some of the stablytransfected cell lines and is compared with superoxide generation by theuntransfected NIH 3T3 cell lysates. The luminescent signal is inhibitedby superoxide dismutase and the general flavoprotein inhibitordiphenylene iodonium, but is unaffected by added recombinant humanp47phox, p67phox and Rac1(GTP-γS), which are essential cytosolic factorsfor the phagocyte respiratory-burst oxidase.

In an alternate and preferred embodiment of the present invention, cellsthat are stably transfected with Nox 4 (YA28), Nox 5 (YA28) or withempty vector (NEF2) are grown in 10 cm tissue culture plates in mediumcontaining DMEM, 10% calf serum, 100 units/ml penicillin, 100 μg/mlstreptomycin, and 1 μg/ml puromycin to approximately 80% confluency.Cells (five tissue culture plates of each cell type) are washed brieflywith 5 ml phosphate buffered saline (PBS) then dissociated from theplates with PBS containing 5 mM EDTA. Cells are pelleted by centrifugingbriefly at 1000×g.

To permeabilize the cells, freeze thaw lysis is carried out and this isfollowed by passage of the cell material through a small bore needle.The supernatant is removed and the cells are frozen on dry ice for 15minutes. After cells are thawed, 200 μl lysis buffer (HANKS' BufferedSalt Solution—HBBS) containing a mixture of protease inhibitors fromSigma (Catalog no. P2714) is added. Cells on ice are passed through an18 gauge needle 10 times and 200 μl of HBSS buffer containing 34%sucrose was added to yield a final concentration of 17% sucrose. Sucroseappeared to enhance stability upon storage. The combination offreeze-thawing and passage through a needle results in lysis ofessentially all of the cells, and this material is referred to as thecell lysate.

The cell lysates are assayed for protein concentration using the BioRadprotein assay system. Cell lysates are assayed for NADPH-dependentchemiluminescence by combining HBSS buffer, arachidonic acid, and 0.01–1μg protein in assay plates (96 well plastic plates). The reaction isinitiated by adding 1.5 mM NADPH and 75 μM lucigenin to the assay mix togive a final concentration of 200 μM NADPH and 10 μM lucigenin, and thechemiluminescence is monitored immediately. The final assay volume as150 μl. The optimal arachidonic acid concentration is between 50–100 μM.A Packard Lumicount luminometer is used to measure chemiluminescence ofthe reaction between lucigenin and superoxide at 37° C. The plate ismonitored continuously for 60 minutes and the maximal relativeluminescence unit (RLU) value for each sample is used for the graph.

The presence of NaCl or KCl within a concentration range of 50–150 μM isimportant for optimal activity. MgCl₂ (1–5 mM) further enhanced activityby about 2-fold. This cell-free assay for Nox 4 NALPH-oxidase activityand the cell-free assay for Nox 5 NADPH-oxidase are useful for screeningmodulators (inhibitors or stimulators) of the Nox 4 enzyme and Nox 5enzyme. The assay may also be used to detect Nox NADPH-oxidase activityin general and to screen for modulators (inhibitors or stimulators) ofthe Nox family of enzymes.

EXAMPLE 10

Nitro Blue Tetrazolium Reduction by Superoxide Generated by NIH 3T3Cells Transfected with the Nox 4 cDNA (SEQ ID NO:1) or the Nox 5 cDNA(SEQ ID NO:3)

Superoxide generation by intact cells is monitored by using superoxidedismutase-sensitive reduction of nitroblue tetrazolium. NEF2 (vectoralone control), YA26 (Ndx 4 (SEQ ID NO:1)-transfected), YA26 (Nox 5 (SEQID NO:3)-transfected), YA28 (Nox 4 (SEQ ID NO:1)-transfected) and YA28(Nox 5 (SEQ ID NO:3)-transfected) cells are plated in six well plates at500,000 cells per well. About 24 hours later, medium is removed fromcells and the cells are washed once with 1 mL Hanks solution (Sigma, St.Louis, Mo.). About 1 mL of filtered 0.25% Nitro blue tetrazolium (NBT,Sigma) is added in Hanks without or with 600 units of superoxidedismutase (Sigma) and cells are incubated at 37° C. in the presence of5% CO₂. After 8 minutes the cells are scraped and pelleted at more than10,000 g. The pellet is re-suspended in 1 mL of pyridine (Sigma) andheated for 10 minutes at 100° C. to solubilize the reduced NBT. Theconcentration of reduced NBT is determined by measuring the absorbanceat 510 nm, using an extinction coefficient of 11,000 M⁻¹cm⁻¹. Some wellsare untreated and used to determine cell number. Because superoxidedismutase is not likely to penetrate cells, superoxide must be generatedextracellularly. The amount of superoxide generated by these cells isabout 5–10% of that generated by activated human neutrophils.

EXAMPLE 11

Modification of Intracellular Components in Nox 4 and Nox 5 TransfectedCells

To test whether superoxide generated by Nox 4 or Nox 5 can affectintracellular targets, aconitase activity in control and Nox-transfectedcell lines is monitored using a method as described in Suh et al. (1999)Nature 401, 79–82. Aconitase contains a four-iron-sulphur cluster thatis highly susceptible to modification by superoxide, resulting in a lossof activity, and has been used as a reporter of intra-cellularsuperoxide generation. Acotinase activity is determined as described inGardner et al. (1995) J. Biol. Chem. 270, 13399–13405. Acotinaseactivity is significantly diminished in the Nox-transfected cell linesdesignated YA26, and YA28 as compared to the transfected control.

Approximately 50% of the aconitase in these cells is mitochondrial,based on differential centrifugation, and the cytosolic andmitochondrial forms were both affected. Control cytosolic andmitochondrial enzymes that do not contain iron-sulfur centers are notaffected. Superoxide generated in Nox 4-transfected cells and Nox5-transfected cells is therefore capable of reacting with and modifyingintracellular components.

EXAMPLE 12

Tumor Generation in Nude Mice Receiving Cells Transfected with the HumanNox 4 cDNA (SEQ ID NO:1) or the Human Nox 5 cDNA (SEQ ID NO:3)

About 2×10⁶ NIH 3T3 cells (either Nox 4-transfected with SEQ ID NO:1,Nox 5-transfected with SEQ ID NO:3, or cells transfected using emptyvector) are injected subdermally into the lateral aspect of the neck of4–5 week old nude mice. Three to six mice are injected for each of threeNox 4-transfected cell lines, each of the Nox 5-transfected cell lines,and 3 mice are injected with the cells transfected with empty vector(control). After 2 to 3 weeks, mice are sacrificed. The tumors are fixedin 10% formalin and characterized by histological analysis.

In another study, 15 mice are injected with Nox 4-transfected NIH 3T3cells. Of the 15 mice injected, 14 show large tumors within 17 days ofinjection, and tumors show expression of Nox 4 mRNA.

In another study, 15 mice are injected with Nox 5-transfected NIH 3T3cells. Of the 15 mice injected, 14 show large tumors within 17 days ofinjection, and tumors show expression of Nox 5 mRNA.

EXAMPLE 13

Demonstration of the Role of Nox 4 and Nox 5 in Non-Cancerous Growth

A role for Nox 4 and Nox 5 in normal growth is demonstrated in rataortic vascular smooth-muscle cells by using antisense to Nox 4 or Nox5. Transfection with the antisense DNA results in a decrease in bothsuperoxide generation and serum-dependent growth. Nox 4 and Nox 5 aretherefore implicated in normal growth in this cell type.

EXAMPLE 14

Expression of Human Nox 4 Protein (SEQ ID NO:2) and Human Nox 5 Protein(SEQ ID NO:4) in a Baculovirus Expression System

SEQ ID NO:2 and SEQ ID NO:4 are also expressed in insect cells usingrecombinant baculovirus. To establish the Nox 4 and Nox 5 expressingvirus systems, the Nox 4 gene (SEQ ID NO:1) or the Nox 5 gene (SEQ IDNO:5) is initially cloned separately into the pBacPAK8 vector (Clontech,Palo Alto, Calif.) and recombinant baculovirus is constructed usingstandard methods according, to manufacturer's protocols. Briefly, PCRamplified Nox 4 DNA or Nox 5 DNA is cloned into the KpnI and EcoRI siteof the vector. Primers used for PCR amplification are SEQ ID NOs:21, 22,23 and 24. Sf9 insect cells (2×10⁶ cells) are infected with 0.5 mg oflinearized baculovirus DNA sold under the trademark BACULOGOLD®(PharMingen, San Diego, Calif.) and 5 mg pBacPAC8 Nox 4 usingTransfection Buffers A and B (PharMingen, San Diego, Calif.). After 5days, the supernatants containing recombinant viruses are harvested andamplified by infecting fresh sf9 cells for 7 days. Amplification iscarried out three times and the presence of the recombinant virusescontaining Nox 4 DNA or Nox 5 DNA is confirmed by PCR using the sameprimers. After three times amplification of viruses, plaque purificationare carried out to obtain the high titer viruses. Approximately 2×10⁸sf9 cells in agar plates are infected for 5 days with serial dilutionsof virus and are dyed with neutral red for easy detection of virusplaques. Selected virus plaques are extracted and the presence of thehuman Nox 4 DNA or human Nox 5 DNA is confirmed again by PCR.

EXAMPLE 15

Antibodies to Human Nox 4 (SEQ ID NO:2) and Human Nox 5 (SEQ ID NO:4)

Polyclonal antibodies are raised separately in rabbits against human Nox4 (SEQ ID NO:2) or human Nox. 5 (SEQ ID NO: 4). Proteins are separatelyconjugated to keyhole limpet hemocyanin (KLH) using glutaraldehyde.

Antigens are injected into different rabbits initially in completeFreund's adjuvant, and are boosted 4 times with antigen in incompleteFreund's adjuvant at intervals of every three weeks. Approximately 0.5mg to 1 mg of peptide is administered at each injection. Blood is drawn1 week after each boost and a terminal bleed is carried out 2 weeksafter the final boost. Anti Nox 4 and anti Nox 5 antibodies are purifiedon affinity columns to which are bound Nox 4 or Nox 5 using techniquesknown to one of ordinary skill in the art. Unbound protein is washedaway with 20 ml of buffer. Elution of the antibodies from the gel wasperformed with 6 ml of elution buffer (100 mM glycine/HCl, pH 2.5, 200mM NaCl, and 0.5% Triton X-100). The eluate is then neutralized byadding 0.9 ml of 1 M Tris/HCl, pH 8.0.

The detection of antigens is performed using an enhancedchemiluminescence kit (Amersham, Buckinghamshire, UK). The affinitypurified antibodies to Nox 4 or to Nox 5 are used at a dilution of1:1000 in a Western blot in which a total of 10 μg of protein is addedto each lane.

EXAMPLE 16

Construction of a Reporter Construct for Nox-1

pGL3-basic (Promega, Madison, Wis.) was used as the parent vector. ThepGL3-basic vector lacks eukaryotic promoter and enhancer sequences,allowing for maximum flexibility in cloning putative regulatorysequences. Expression of luciferase activity in cells transfected withthis plasmid depends on insertion and proper orientation of a functionalpromoter upstream from luc+. Potential enhancer elements can also beinserted upstream of the promoter or in the BamH I or Sal I sitesdownstream of the luc+ gene. Primers SEQ ID NO: 25,5′GCTACTCGAGTGTGCCAATTTCACCTGGCAT-3′ and SEQ ID NO:26,5′-AACTCTCGAGTGTCAAGAGGTGGTTTGGAGC-3′ were used along with genomic DNAto obtain the promoter region of Nox 1 (SEQ ID NO:5) flanked by Xhorestriction sites. The restriction sites were then used to insert theNox 1 promoter region into the pGL3 plasmid. (See FIG. 7). Successfultransfection was determined by the activity of luciferase which wasmeasured using a luminometer.

EXAMPLE 17

Use of the Reporter Construct as an Assay

The construct from Example 16 is stably transfected into human Caco-2 orHT-29 cells. Transfection is carried out as described in Sambrook etal., Molecular Cloning, A Laboratory Manual, Volumes 1–3, 2nd edition,Cold Spring Harbor Laboratory Press, N.Y., 1989. The SEQ ID NO:5 inpEF-PAC and the empty vector are separately transfected into Caco-2cells using Fugene 6 (Boeringer Mannheim). 10⁵ to 10³ cells stablytransfected with human Nox 1 promoter gene SEQ ID NO:5 and with emptyvector are prepared in 0.3% warm (40° C.) agar solution containing DMEMand 10% calf serum. Cells are distributed onto a hardened 0.6% agarplate prepared with DMEM and 10% calf serum. After three weeks inculture (37° C., 5% CO₂) colony formation is observed by microscopy.About 2×10⁶ cells maintained in DMEM containing 10% calf serum aretransfected with 10 μg of DNA. After 2 days, cells are split andselected in the same medium containing 1 mg/ml puromycin. Colonies thatsurvive in selection media for 10 to 14 days are subculturedcontinuously in the presence of puromycin.

The colonies are used as a screening assay by adding compounds to themedia suspected of effecting the expression of ROI. Measurement of theluciferase output indicates whether a compound enhanced or inhibited theinduction of the Nox 1 gene and facilitates the development of drugsbased on a compound's cellular effects.

EXAMPLE 18

Expression of Nox 3, Nox 4, and Nox 5 mRNA in Cancer Cells

Many cancer cells overproduce reactive oxygen species (Szatrowski andNathan, 1991), and this may be causative in the transformed phenotype(Suh et al., 1999). The expression of Nox 1–5 was investigated in avariety of human tumor and other cell lines, to determine if theseenzymes might account for reactive oxygen generation seen in sometumors. Expression of Nox family members in human cancers. RT-PCR wascarried out as in FIG. 5. FIG. 6A shows Nox expression in the followingcell lines: ES-2 (ovarian clear cell carcinoma), PA-1 (ovarianteratocarcinoma), Ovcar-3 (ovarian adenocarcinoma), MDA-MB-231 (mammaryadenocarcinoma), SKO-007 (plasmacytoma), Caco-2 (colon carcinoma), T84(colon carcinoma), HEK293 (embryonic kidney transformed withadenovirus), and Hela (cervical adenocarcinoma). FIG. 6B show Noxexpression in five cell lines derived from human glioblastomas, as wellas from human astrocyte primary cultures. FIG. 6C shows the ratio ofexpression of gp91phox, Nox 4 and Nox 5 compared with G3PDH, obtainedfrom real time PCR results.

As shown in FIGS. 6A and 6B, Nox isoforms were expressed in 12 out ofthe 14 tumor or transformed cell lines examined. Nox 1 is expressed intwo colon cancer lines, Caco-2 and T-84, as well as in the transformedcell line HEK293, and to a lesser extent in Hela cells. Nox 4 was seenin 11 of these cell lines, while Nox 5 was seen in 7. gp91phox was alsoexpressed in more than half of the cell lines. The identity of the mRNAswas confirmed by sequencing as indicated in FIGS. 5, 6A and B.

In live brain tumor cell lines derived from human glioblastomas, Nox 4was always expressed, along with variable expression of Nox 5 andgp91phox (FIG. 6B). Real time PCR revealed that the ratio of expressionof Nox to G3PDH varied significantly in the various tumor cell linescompared with primary human astocytes (FIG. 6C). Although the cellularorigin of glioblastomas has not been definitively established, thiscancer type is thought by many workers to have arisen from theastrocytic lineage.

The expression of Nox forms in cancer and transformed cell lines did notcorrelate strictly with the expression in normal tissue, indicating thatexpression of Nox isoforms is sometimes altered in cancer cells. Thus,aberrant expression or regulation of Nox isoforms could account for theincreased reactive oxygen generation seen in some cancer cells.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. U.S. provisional patentapplications Ser. Nos. 60/249,305, 60/251,364, 60/289,172, 60/289,537are hereby incorporated by reference in their entirety. It should beunderstood that the foregoing relates only to preferred embodiments ofthe present invention and that numerous modifications or alterations maybe made therein without departing from the spirit and the scope of thepresent invention as defined in the following claims.

1. A method of determining the activity of an agent to decrease cell division, comprising contacting a protein comprising the amino acid sequence of SEQ ID NO:4, or a fragment of SEQ ID NO: 4 capable of superoxide production, or a substitution thereof of less than 1% of the amino acid sequence, with the agent; and measuring the ability of the agent to decrease superoxide production, inhibit diaphorase activity or bind the protein as compared to a control not contacted with the agent, thereby determining the ability of the agent to decrease cell division.
 2. The method of claim 1, wherein the protein comprises the amino acid sequence of SEQ ID NO:4 or a fragment of SEQ ID NO: 4 capable of superoxide production.
 3. The method of claim 1, wherein the method comprises measuring the ability of the agent to inhibit superoxide production.
 4. The method of claim 1, wherein the method comprises measuring the ability of the agent to bind the protein.
 5. The method of claim 1, wherein the protein comprises the C-terminal 265 amino acids of SEQ ID NO:
 4. 6. The method of claim 1, wherein the method comprises measuring the ability of the agent to inhibit diaphorase activity.
 7. The method of claim 6, wherein measuring the ability of the agent to inhibit the diaphorase activity comprises evaluating NAPDH-dependent reductase activity.
 8. The method of claim 6, wherein evaluating the diaphorase activity of the protein comprises use of cytochrome c or nitrobluetetrazolium.
 9. A method for identifying an agent that modulates the activity of a Nox enzyme, comprising contacting an agent of interest with a protein comprising the C-terminal 265 amino acids of SEQ ID NO: 4 capable of superoxide production; and evaluating the diaphorase activity of the protein; wherein a decrease in the diaphorase activity of the protein as compared to a control indicates that the agent modulates the activity of a Nox enzyme.
 10. The method of claim 9, wherein evaluating the diaphorase activity of the protein comprises evaluating NAPDH-dependent reductase activity.
 11. The method of claim 9, wherein evaluating the diaphorase activity of the protein comprises use of cytochrorue c or nitrobluetetrazolium. 